Project 2: Project Summary/Abstract Broadly neutralizing antibodies (bNAbs) against HIV-1 can inhibit viral replication by two distinct mechanisms. One is the neutralization of virus, which prevents infection. The other is its ability to kill HIV-1 infected cells through activation of complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Thus, bNAbs have the potential to not only inhibit new viral infection cycles but also eliminate chronically infected cells, which are necessary for cure of the HIV-1 disease. However, the serum concentrations of the antibodies must be kept at therapeutic levels for long periods of time to eliminate HIV-1-infected cells, because persistently infected cells reside in the deep tissue. Therefore, multiple administrations of the antibodies are required, which could be expensive and labor-intensive. In addition, administration of recombinant bNAbs can raise host immune reactions to the bNAbs, leading to loss of neutralization and cytotoxicity activities of bNAbs and shortening their half-life. Gene therapy vectors, especially lentiviral/oncoretroviral vectors that integrate their transgene into host chromosomes, can produce bNAbs in vivo for long periods. But immune reactions against the bNAbs can still decrease the antiviral efficacy of bNAbs and shorten the duration of bNAb expression. B- cells physiologically express wide varieties of valuable antibody regions generated by recombination and mutations in genes. B-cells are known to induce tolerance to the valuable regions. This ability of B-cells was previously used to induce tolerance to self-antigens for therapy of autoimmune diseases and coagulation factor IX for treatment of hemophilia. Therefore, transduction of B-cells with bNAb expressing vectors is likely to generate long-term bNAbs without inducing immune reactions to the bNAbs. We have developed lentiviral vectors that can specifically transduce desired target cell types after systemic administration. By specific transduction of B-cells by this lentiviral vector, we will attempt to express bNAbs for long periods of time by avoiding immune reactions against bNAbs. We will also attempt to prolong the duration of bNAb expression by differentiating transduced B-cells to long-lived plasma cells and memory B-cells, which have long life-span. We will then investigate if bNAbs expressed from B-cells can eliminate HIV-1-infected cells in humanized BLT mice. Since commonly used humanized BLT mice do not have normal complement, we will use a novel type of humanized BLT mice that have intact complement system for full CDC activity of the bNAb. We will next investigate whether bNAbs expressed from B-cells and T-cells engineered to express anti-HIV-1 transgene can synergistically eliminate HIV-1 infected cells in the humanized B-cells. Lastly, we will investigate whether systemic administration of our B-cell targeting lentiviral vector can specifically transduce B-cells in non-human primates (NHP) by analyzing biodistribution and cell types of transduced cells. We will also investigate the pharmacokinetics of and immune reactions to bNAbs expressed in NHP. These experiments are designed to develop a novel bNAb-based gene therapeutic approach, which can be applicable to other infectious diseases.